Biology subjects

Dorn, T.

Publications and source records attributed to Dorn, T..

4 recordsLinked to original sources

bioRxiv2026

A new bioorthogonal PET reporter gene for cardiac regenerative medicine binding to DTPA-lanthanide complexes

The lack of non-invasive, quantitative methods to track the location and proliferation of transplanted cells hampers the clinical translation of cardiac regenerative therapies. Here, we establish a PET reporter for tracking human induced pluripotent stem cell (hiPSC)-derived cardiac cells across complementary two-dimensional (2D) and three-dimensional (3D) cardiac tissue models. Methods hiPSCs were genetically engineered using CRISPR-Cas9 to express a human-derived, anticalin-based, bioorthogonal PET reporter (DTPA-R) that specifically binds radiolabeled DTPA-metal complexes. The engineered hiPSCs were differentiated into ventricular progenitors (HVPs) and cardiomyocytes (CMs). Stability of reporter gene expression and its effects on cellular differentiation and function were evaluated. Uptake of [18F]F-DTPA*Tb by DTPA-R-positive and -negative HVPs was analyzed in vitro, and DTPA-R HVPs were evaluated in ex vivo cultured porcine myocardial slices with and without radiofrequency ablation (RFA) injury. Phantom studies were performed to evaluate PET detectability under clinically relevant large-animal and human imaging conditions and to assess how activity distribution affects signal detection in relation to DTPA-R cardiac cell-equivalent numbers. Results DTPA-R expression was maintained during the differentiation stages of hiPSC-derived cardiac cells and had no measurable impact on migration, differentiation, and functional integration. PET imaging enabled the specific detection of DTPA-R HVPs in intact and injured myocardial slices. At the initial phantom scan, the measured activity corresponded to cell-equivalent numbers of approximately 3.2 x 10^6 HVPs or 5.5 x 10^6 CMs. The compact 200 uL source remained clearly detectable at 6 h, whereas the more dispersed 1,000 uL source progressively approached background activity, indicating that PET detectability depended strongly on local activity concentration and distribution. Conclusion This study establishes a bioorthogonal PET reporter gene platform for quantitative imaging of hiPSC-derived cardiac cells, with no measurable adverse effects on the biological parameters examined, and provides a methodological foundation for future translational and in vivo imaging studies. The approach may also be applicable to other hiPSC-derived cell products beyond cardiac cell lineages.

bioengineering↗

bioRxiv2022

Retinoic acid signaling modulation guides in vitro specification of human heart field-specific progenitor pools

Cardiogenesis relies on the precise spatiotemporal coordination of multiple progenitor populations. Understanding the specification and differentiation of these distinct progenitor pools during human embryonic development is crucial for advancing our knowledge of congenital cardiac malformations and designing new regenerative therapies. By combining genetic labelling, single-cell transcriptomics, and ex vivo human-mouse embryonic chimeras we uncovered that modulation of retinoic acid signaling instructs human pluripotent stem cells to form heart field-specific progenitors with distinct fate potentials. In addition to the classical first and second heart fields, we observed the appearance of juxta-cardiac field progenitors giving rise to both myocardial and epicardial cells. Applying these findings to stem-cell based disease modelling we identified specific transcriptional dysregulation in first and second heart field progenitors derived from stem cells of a patient with hypoplastic left heart syndrome. This highlights the suitability of our in vitro differentiation platform for studying human cardiac development and disease.

cell biology↗

bioRxiv2021

Sequential defects in cardiac lineage commitment and maturation cause hypoplastic left heart syndrome

BackgroundComplex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role. MethodsTo identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed: i) whole-exome sequencing of 87 HLHS parent-offspring trios, ii) nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, iii) single cell RNA sequencing and iv) 3D modeling in iPSCs from 3 patients with HLHS and 3 controls. ResultsGene set enrichment and protein network analyses of damaging de-novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell-cycle and cardiomyocyte maturation. Single-cell and 3D modeling with iPSCs demonstrated intrinsic defects in the cell-cycle/UPR/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte-subtype differentiation/maturation in HLHS. Additionally, premature cell-cycle exit of ventricular cardiomyocytes from HLHS patients prevented normal tissue responses to developmental signals for growth leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues. ConclusionsOur results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

developmental biology↗

bioRxiv2020

Primate heart regeneration via migration and fibroblast repulsion by human heart progenitors

SUMMARYHuman heart regeneration is one of the most critical unmet clinical needs at a global level1. Muscular regeneration is hampered both by the limited renewing capacity of adult cardiomyocytes2-4 and the onset of cardiac fibrosis5,6, resulting in reduced compliance of the tissue. Primate have proven to be ideal models for pluripotent stem cell strategies for heart regeneration, but unravelling specific approaches to drive cell migration to the site of injury and inhibition of subsequent fibrosis have been elusive. Herein, by combining human cardiac progenitor lineage tracing and single-cell transcriptomics in injured non-human primate heart bio-mimics, we uncover the coordinated muscular regeneration of the primate heart via directed migration of human ventricular progenitors to sites of injury, subsequent fibroblast repulsion targeting fibrosis, and ultimate functional replacement of damaged cardiac muscle by differentiation and electromechanical integration. Single-cell RNAseq captured distinct modes of action, uncovering chemoattraction mediated by CXCL12/CXCR4 signalling and fibroblast repulsion regulated by SLIT2/ROBO1 guidance in organizing cytoskeletal dynamics. Moreover, transplantation of human cardiac progenitors into hypo-immunogenic CAG-LEA29Y transgenic porcine hearts following injury proved their chemotactic response and their ability to generate a remuscularized scar without the risk of arrhythmogenesis in vivo. Our study demonstrates that inherent developmental programs within cardiac progenitors are sequentially activated in the context of disease, allowing the cells to sense and counteract injury. As such, they may represent an ideal bio-therapeutic for functional heart rejuvenation.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗